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UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS 

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Vol.  11,  No.  4,  pp.  53-88,  pi.  1  March  31,  1913 


THE  CONTROL  OF  PIGMENT  FORMATION 
IN  AMPHIBIAN  LARVAE 


BY 

MYRTLE  E.  JOHNSON 


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UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

ZOOLOGY 

Vol.  11,  No.  4,  pp.  53-88,  pi.  1  March  31,  1913 


THE   CONTROL  OF  PIGMENT  FORMATION 
IN  AMPHIBIAN  LARVAE* 

BY 

MYRTLE  E.  JOHNSON 


CONTENTS 

PAGE 

A.  Introduction    53 

B.  Current  theories  of  pigment  formation  54 

C.  The  relation  of  amount  of  nutrition  to  pigmentation  '. 59 

D.  Effect  of  various  kinds  of  foods  upon  pigmentation  64 

E.  Effect  of  lecithin  and  various  foods  used  in  experimentation,  upon 

the  tyrosinase  reaction  69 

F.  Effect  of  lecithin  upon  pigmentation  73 

G.  Effect  of  certain  other  organic  substances  upon  the  tyrosinase  re- 

action and  upon  pigmentation  76 

H.  Histological  differences  between  chromatophores  of  larvae  fed  upon 

different  foods  80 

I.  Effect  of  changes  in  light,  heat,  and  food  upon  pigmentation  82 

J.  Summary  83 

K.  Bibliography  :. 84 


A.  INTRODUCTION 

The  experiments  considered  in  this  paper  grew  out  of  a  series 
of  feeding  experiments  carried  on  with  larvae  of  Hyla  regilla 
and  Rana  sp.  While  size  differences  only  were  considered  at 
first,  color  differences  soon  became  so  marked  as  to  demand 
attention.  The  amount  of  black  pigment  in  the  different  lots  of 
tadpoles  varied  considerably  and  this  variation  was  not  correlated 
with  the  size  of  the  tadpole.  It  was  apparent  that  certain  foods 


*  A  thesis  presented  to  the  Faculty  of  the  College  of  Natural  Sciences, 
in  the  University  of  California,  in  partial  satisfaction  of  the  requirementa 
for  the  degree  of  Doctor  of  Philosophy.  April,  1912. 


54  University  of  California  Publications  in  Zoology    [VOL.  11 

contain  elements  which  govern  the  formation  of  pigment  inde- 
pendently of  the  effect  of  the  food  upon  the  size  of  the  organism. 

It  was  found  that  most  of  the  tadpoles  showed  a  large  or 
medium  amount  of  pigment  excepting  those  that  were  fed  on 
yolk  of  egg,  which  showed  a  much  smaller  amount.  Experi- 
ments showed  that  when  lecithin  was  fed  along  with  foods  which 
ordinarily  produced  much  black  pigment  that  a  much  smaller 
amount  of  pigment  was  produced.  Experiments  with  tyrosinase 
showed  that  the  tyrosinase  reaction  could  in  a  measure  be  in- 
hibited by  the  addition  of  lecithin  or  the  products  of  digestion 
of  egg  yolk.  The  experiments  thus  give  an  example  of  a  chemical 
substance  inhibiting  the  tyrosinase  reaction  and  when  fed  to  the 
tadpoles  inhibiting  to  some  extent  the  production  of  melanin 
pigment  in  the  epidermis. 

This  investigation  has  been  carried  on  in  the  zoological  lab- 
oratory of  the  University  of  California  under  the  direction  of 
Professor  Harry  Beal  Torrey,  and  I  am  greatly  indebted  to  him 
for  his  continued  help  and  encouragement.  I  also  wish  to  express 
to  Professors  T.  B.  Robertson  and  H.  C.  Biddle  of  this  Univer- 
sity my  thanks  for  their  kind  interest  and  assistance  in  questions 
of  physiological  and  organic  chemistry. 

B.  CURRENT  THEORIES  OF  PIGMENT  FORMATION 

In  relating  these  results  to  the  current  theories  of  color  for- 
mation and  inheritance  it  will  be  convenient  to  consider  Weis- 
mann  's  germinal  selection  theory,  aspects  of  the  Mendelian  theory 
of  inheritance,  and  the  results  of  recent  biochemical  investiga- 
tion. 

Weismann  postulates  determinants,  aggregations  of  ultimate 
1  (  vital  units  '  '  capable  of  transmission  through  the  germ  cells  and 
able  to  determine  "  hereditary  characters"  of  the  body.  Their 
presence  determines  the  specific  development  of  a  particular  part 
of  the  body  which  may  consist  of  a  group  of  cells,  a  single  cell 
or  a  part  of  a  cell.  A  struggle  for  existence  between  the  biophores, 
the  smallest  elements  composing  the  determinants,  continues 
throughout  the  development  of  the  embryo,  different  biophores 
being  able  to  appropriate  different  amounts  or  kinds  of  nourish- 
ment. These  differences  in  nourishment  cause  inequalities  in  the 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         55 

growth  of  the  different  biophores  which  produce  differences  in 
the  constitution  of  the  determinants  and  consequently  qualitative 
variations  in  the  organism.  These  variations  may  be  spon- 
taneous resulting  from  intra-germinal  nutritive  conditions  and 
therefore  not  affecting  all  the  ids  at  once  or  they  may  be  brought 
about  by  extra-germinal  influences  affecting  all  the  ids  at  the 
same  time. 

Among  Mendelians  the  factor  hypothesis  plays  an  important 
role.  Castle  (1909)  names  the  following  color  factors  which  he 
finds  in  the  case  of  the  gray  rabbit : 

"Symbol  C.  A  common  color  factor  necessary  to  the  production  of 
all  pigment,  wanting  only  in  albinos. 

' '  B.  A  factor  for  black,  some  substance  which  acting  upon  C 
produces  black  pigmentation. 

"  Br.  A  factor  for  brown,  some  substance  which  acting  upon 
C  produces  a  chocolate-brown  pigmentation. 

"  Y.  A  factor  for  yellow,  some  substance  which  acting  upon 
C  produces  yellow  pigmentation. 

11  I.  An  intensity  factor,  which  determines  whether  the 
pigmentation  shall  be  intense  (as  in  black  and  in 
yellow),  dilute  (as  in  blue  and  in  cream),  or  of  some 
intermediate  degree  of  intensity. 

"  A.  A  pattern  factor  which  causes  the  black  or  brown  pig- 
ments to  be  excluded  from  certain  portions  of  the 
individual  hairs,  where  yellow  then  shows.  A 
' '  ticked ' '  gray  coat  results.  When  this  factor  is 
present  the  under  surfaces  of  the  rabbit  (tail,  belly) 
are  unpigmented  (white).  The  symbol,  A,  stands  for 
agouti,  this  factor  having  first  been  demonstrated  in 
the  "agouti"  guinea-pig.  (See  Castle,  1907.) 

"  U.  A  factor  for  uniformity  of  pigmentation  (in  distinction 
from  spotting  with  white,  S). 

"  E.  A  factor  governing  the  extension  of  black  and  of  brown 
pigmentation,  but  not  of  yellow.  When  most  re- 
stricted in  distribution  the  black  or  brown  pigments 
are  found  in  the  eye  and  in  the  skin  of  the  extremities 
only,  but  not  in  the  hair,  when  more  extended  they 
occur  also  in  the  hair  generally." 

The  various  types  of  coloration  seen  in  different  rabbits  are 
represented  by  means  of  various  arrangements  of  these  symbols 
—in  fashion  resembling  the  formulae  for  the  constitution  of 
molecules  of  organic  compounds. 


56  University  of  California  Publications  in  Zoology    [VOL.  11 

As  to  the  form  and  composition  of  these  Mendelian  factors, 
Castle  says  we  can  at  present  give  no  satisfactory  answer,  but  he 
adds  (p.  68)  :  "It  is,  however,  we  think,  not  necessary  to  suppose 
that  there  exist  in  the  minute  germ-cell  as  many  complex  organic 
substances  as  there  are  activities  of  the  cell ;  neither  is  it  necessary 
to  suppose  a  different  substance  present  for  every  independent 
factor  identified.  The  various  independent  factors  may  have  a 
basis  no  more  complicated  than  that  of  so  many  atoms  attached  to 
a  complex  molecular  structure.  Experiment  shows  that  the  factors 
may  be  detached  one  by  one  from  the  organic  complex.  The 
discontinuity  of  their  coming  and  going  is  entirely  in  harmony 
with  the  conception  of  them  as  components  merely  of  complex 
molecular  bodies."  Such  a  view  attempts  to  provide  for  segre- 
gation of  characters  without  discussing  vital  units. 

Among  the  chemists  is  Dewitz  (1902)  who  experimented  with 
fly  larvae  (Lucilia  Caesar)  and  found  that  while  there  was  no 
tyrosinase  in  very  young  larvae  (one  or  two  days  old)  older 
larvae  contained  a  considerable  quantity.  When  the  pupae 
formed  they  rapidly  became  pigmented,  but  this  pigmentation 
could  not  go  on  without  oxygen. 

Phisalix  (1905)  working  with  cockroach  larvae,  found  that 
the  larvae  when  hatched  were  colorless,  but  within  three  hours 
changed  through  grey  and  brown  to  black.  Phisalix  concludes 
that  tyrosin  and  tyrosinase  exist  in  the  embryo  long  before  the 
color  develops  and  that  "it  is  probable  that  they  coexist  in  the 
egg  or  that  they  are  deposited  at  the  time  of  ovogenesis. ' ' 

Roques  (1909)  found  that  during  the  metamorphosis  of 
Limnophilus  flavicornis  Fabr.,  the  amount  of  tyrosinase  in  the 
body  was  greatest  just  before  pigmentation  began  and  as  the 
amount  of  pigment  increased,  the  tyrosinase  decreased,  until  it 
was  entirely  absent  when  the  beetle  was  fully  pigmented. 

Riddle  (1909,  p.  329)  reviews  our  knowledge  of  melanin 
color  formation  and  urges  the  futility  of  piling  up  factors  one 
for  every  color  in  the  germ  to  explain  the  production  of 
melaninic  color  since  without  them  "  in  an  animal  that  pro- 
duces melaninic  color,  there  exists  all  the  machinery  necessary 
to  produce  a  series  or  scale  of  these  colors."  He  continues  (p. 
336)  :  "This  means  that  the  animal  that  transmits  the  enzyme 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         57 

for  black,  i.e.,  produces  black-colored  offspring  must  at  the  same 
time  transmit  also  the  enzyme  for  brown,  chocolate,  red,  yellow, 
etc.  (more  accurately,  an  enzyme  for  each  step  of  oxidation  from 
tyrosin  to  black  melanin),  without  the  absence  of  a  single  one." 
In  a  footnote  (p.  337)  he  adds:  "The  sort  of  specificity  of 
enzymes  that  has  thus  far  been  assumed  by  the  Mendelians  has, 
however,  been  of  a  different  sort;  namely,  that  for  the  produc- 
tion of  each  color  only  one  enzyme  is  necessary,  but  the  enzyme 
which  produces  any  particular  color  is  specifically  different  from 
those  which  produce  other  colors;  .  .  .  ." 

Riddle  (1909,  p.  336)  maintains  that  with  tyrosinase  able  to 
produce  a  series  of  colors,  several  of  Castle's  factors  (A,  B,  E, 
I,  and  D)  may  be  reduced  to  one,  tyrosinase,  while  C,  the 
chromogen,  may  be  left  out  entirely  as  a  factor  since  such  a 
chromogen  is  "universal  in  protoplasm."  In  conclusion  he  asks: 
"Is  it  too  much  to  expect  that  the  further  application  of  such 
tests  as  the  one  here  presented  in  outline  for  the  melanin  colors 
will  in  the  end  remove  many  of  the  Mendelian  *  factors'  from 
the  germ  cells  ?  That  many  of  their  '  characters '  will  come  to  rest 
on  a  more  proximate  basis;  will  be  known  to  have  their  'deter- 
mination' and  origin  in  very  general  germinal  powers,  and  in 
somatic  conditions  obtaining  previous  to,  or  at  the  time  of,  their 
development  ? ' ' 

Gortner  (1910a,  1911a,  1911c)  has  recently  published  results 
of  similar  investigations  on  the  meal  worm,  cicada,  and  potato 
beetle.  He  finds  that  in  the  meal  worm  the  chromogen  is 
secreted  only  as  needed  for  pigmentation  and  is  present  in  exceed- 
ingly small  amounts  at  any  one  time,  that  tyrosinase  is  present 
in  both  the  pupa  and  beetle,  but  the  chromogen  is  apparently 
lacking  in  the  pupa  stage,  the  only  stage  without  pigmentation. 

In  the  case  of  the  periodical  cicada,  the  tyrosinase  is  not 
found  in  the  body  of  the  pupa  or  the  adult  but  is  apparently 
secreted  with  the  new  cuticula  since  the  oxidase  is  present  in 
water  in  which  the  newly  emerged  adults  have  been  washed. 
In  his  study  of  the  Colorado  potato  beetle  (Leptinotarsa  decem- 
lineata  Say)  Gortner  finds  the  pigmentation  "produced  by  the 
interaction  of  an  oxidizing  enzyme  of  the  tyrosinase  type,  and 
an  oxidizable  chromogen.  The  color  pattern  is  caused  by  the 


58  University  of  California  Publications  in  Zoology    CV°L-  n 

localized  secretion  of  chromogen."  Gortner  (1911d)  after 
further  investigation  of  different  melanins  concludes  that  they 
are  formed  by  the  interaction  of  an  oxidase  and  an  oxidizable 
chromogen.  He  finds  that  there  are  at  least  two  types  of  melanin 
which  may  be  differentiated  by  their  appearance  and  by  their 
solubility  in  acids.  He  thinks  it  probable  that  the  two  types  are 
formed  by  the  oxidation  of  different  chromogens. 

Some  of  these  later  results,  it  seems  to  me,  lead  one  to  take  a 
little  different  view  of  the  situation  from  the  one  taken  by 
Riddle.  He  (1909,  p.  334)  speaks  of  the  different  colors  of  the 
tyrosin  series  as  obtainable  by  different  degrees  of  oxidation,  for 
instance  he  says:  "At  present  the  biological  data  are  wanting  to 
quantitatively  seriate  all  of  the  several  colors ;  but  there  is  appar- 
ently enough  data  to  warrant  the  definite  statement  that  yellow 
mice  are  forms  with  the  power  to  oxidize  tyrosin  compounds 
to  an  intermediate  point."  If  we  adopt  this  view  we  are  under 
the  necessity  of  postulating  some  chemical  difference  between 
the  oxidase  in  yellow  mice  and  that  in  brown  mice  that  causes 
this  difference  in  oxidation. 

The  results  of  Bertrand  (1908)  and  of  Abderhalden  and 
Guggenheim  (1907,  1908)  show  that  the  end  result  of  the  series 
of  colors  shown  by  melanin  pigment  is  different  when  the  tyrosin 
exists  in  different  combinations,  or  when  different  chromogens  are 
used.  Gortner  showed  that  the  spots  on  the  elytra  of  the  potato 
beetle  are  due  to  a  localized  secretion  of  the  chromogen  rather 
than  to  a  difference  in  the  oxidase  which  is  apparently  present 
over  the  whole  area.  It  would  appear  from  these  facts  that 
slight  differences  in  color  between  two  individuals  are  due  to 
slight  differences  in  the  combination  in  which  the  chromogen 
exists  (or  existed  when  the  pigment  was  produced)  and  that 
variations  in  the  marking  of  different  individuals  are  due  to 
slight  variations  in  the  character  or  diffusion  of  the  chromogen 
in  different  localities. 

The  difference  in  color  in  the  case  of  the  tadpoles  here  de- 
scribed seems  to  be  brought  about  by  a  reduction  in  the  amount 
of  black  pigment  rather  than  by  a  change  in  the  color  of  the 
pigment.  This  reduction  in  the  amount  of  black  pigment  allows 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         59 

the  epidermal  pigments  of  other  sorts  and  colors  to  come  into 
greater  prominence. 

One  must  distinguish  then  between  differences  in  coloration 
which  involve  actual  differences  in  the  color  of  one  or  more  of 
the  pigments  and  those  which  involve  differences  in  the  propor- 
tional amounts  of  the  different  pigments.  Although  the  inhibitors 
here  discussed  merely  reduce  the  amount  of  melanin,  instances 
cited  show  that  the  tyrosinase  reaction  may  be  so  affected  by 
certain  chemical  substances  that  the  color  of  the  resulting  pig- 
ment is  different  from  that  of  ordinary  melanin.  The  " factors" 
postulated  by  Mendelians  may  operate  in  this  latter  fashion,  also 
the  chemical  substances  cited  below  which  are  able  to  change  the 
color  of  the  plumage  of  birds  undoubtedly  act  in  this  way. 

C.  THE   EELATION   OF   AMOUNT    OF    NUTRITION    TO 
PIGMENTATION 

Weismann's  view  that  color  is  profoundly  influenced  by 
nutritive  conditions  is  supported  by  Tornier's  experiments  in 
pigment  control.  My  results  with  similar  experiments,  however, 
do  not  support  this  view. 

Tornier  (1907,  1908),  experimenting  with  Pelobates  larvae, 
divided  the  tadpoles  into  lots  for  differential  feeding.  He  found 
that  tadpoles  receiving  a  minimum  amount  of  food  (algae, 
together  with  varying  amounts  of  fish)  contained  little  or  no 
pigment  and  that  by  feeding  increased  amounts  of  fish  it  was 
possible  to  change  the  epidermal  coloring  from  white  through 
yellow,  red,  and  grey,  to  black.  Comparable  effects  were  pro- 
duced by  removing  a  portion  of  the  yolk  from  the  egg.  This 
reduced  the  nutrition  of  the  animals  and  produced  albinism, 
erythrinism,  or  blackness  depending  upon  the  state  of  nutrition. 
He  also  found  that  the  experiments  could  be  carried  in  the 
opposite  direction  and  that  through  a  diminution  of  feeding,  well 
fed  black  larvae  could  be  changed  back  through  a  series  of  colors 
in  the  order  of  black,  brown,  red,  grey,  white.  He  sought  to  show 
not  only  that  increase  in  food  supply  caused  a  greater  develop- 
ment of  pigment  in  the  chromatophores  but  also  that  the  pigment 


60  University  of  California  Publications  in  Zoology    [VOL.  11 

granules  of  the  chromatophores  acted  as  reserve  material  in  cases 
of  inanition. 

Experiments  similar  to  these  of  Tornier  were  carried  on  by 
me  during  two  successive  seasons  with  Hyla  and  Rana  tadpoles. 
The  results  do  not  confirm  those  of  Tornier,  since  the  tadpoles 
receiving  a  small  amount  of  food  were  no  lighter  than  those 
receiving  an  abundance. 

The  tadpoles  from  a  single  egg  mass  were  divided  into  three 
lots  and  each  lot  given  a  different  amount  of  food.  As  it  is 
impossible  when  feeding  graduated  amounts  of  food  to  be  sure 
that  the  tadpoles  in  a  given  dish  share  the  food  alike,  instead  of 
placing  different  amounts  of  food  in  the  different  dishes,  the  food 
was  withheld  from  the  dishes  for  different  lengths  of  time.  On 
days  when  the  tadpoles  received  food  they  were  given  as  much 
as  they  could  eat,  and  on  other  days  all  food  was  removed  from 
the  dishes.  One  set  of  tadpoles  was  fed  every  day,  the  food  of 
the  second  set  was  withheld  every  third  day  and  that  of  the  third 
set  was  withheld  every  second  day.  The  three  sets  of  tadpoles 
thus  received  large,  medium,  and  small  amounts  of  food  approxi- 
mately in  the  ratio  of  1 :  %  :  %. 

The  growth  of  the  tadpoles  was  proportional  to  the  amount 
of  food  received  and  the  amount  of  pigment  seemed  also  to  be 
proportioned  to  size  since  the  large,  medium,  and  small  tadpoles 
on  the  same  diet  were  all  of  the  same  color,  those  that  were  half 
starved  being  no  lighter  than  those  that  were  large  and  well  fed. 
In  order  that  this  might  not  be  a  prejudiced  judgment  the  dif- 
ferent sets  of  tadpoles  were  repeatedly  submitted  to  other 
observers  before  explaining  the  object  of  the  experiment.  The 
verdict  always  was  that  the  tadpoles  were  all  the  same  color  or 
that  the  smaller  ones  were  a  little  darker.  The  experiments  were 
performed  for  two  successive  years  with  the  same  result  (table  1) . 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         61 


TABLE  1 

Length  measurements  and  color  of  tadpoles  fed  on  different  amounts  of 
food.  Tadpoles  of  lots  A  and  B  were  all  the  same  size  when  the  experi- 
ment began  Feb.  10,  1912;  those  of  C  and  D  were  the  same  size 
when  the  experiment  began,  April  28,  1911.  Length  measurements  for 
lots  A  and  B  represent  average  length  of  all  tadpoles  in  the  dish,  those 
for  C  and  D,  the  average  of  several  typical  tadpoles. 


Amount 

of 
food 

Full  amount 
%  full  amount 
y2  full  amount 


A.  Eana  tadpoles  fed  on  liver 
Length  in  mm. 


Feb.  13 
16.1 
15.4 
15.6 


Feb.  20 
21.4 
20.2 
19.1 


Mar.  6 
27.4 
24.0 
22.5 


Number 

of 
tadpoles 


Color 
Dark 
Dark 
Dark 


B.  Eana  tadpoles  fed  on  egg  yolk 


Amount 

of 
food 

Full  amount 
%  full  amount 
1/  full  amount 


Amount 

of 
food 

Full  amount 
%  full  amount 
y2  full  amount 


Length  in  mm. 

Number 
of 
tadpoles 

8 
8 
8 

Color 
Light 
Light 
Light 

Feb.  13 
15.3 
15.0 
15.4 

Feb.  20 
21.3 
19.5 
19.1 

Mar.  6 
28.1 
24.4 
23.2 

C.  Hyla  tadpoles  fed  on  liver 


Length  in  mm. 


May  10 
16 
13 
12 


June  3 
30 
23 
20 


Number 

of 
tadpoles 


Color 
Dark 
Dark 
Dark 


Amount 

of 
food 

Full  amount 
%  full  amount 
y2  full  amount 


D.  Hyla  tadpoles  fed  on  egg  yolk 


Length  in  mm. 

Number 
of 
tadpoles 

4 
4 
5 

Color 
Medium  light 
Medium  light 
Medium  light 

May  10 
20 
14 
13 

June  3 
32 
24 

18 

Owing  to  accidents  and  the  small  allowance  of  food,  most  of 
the  tadpoles  living  on  half  the  regular  fare  died  before  meta- 
morphosing. I  have  one  preserved  specimen,  however,  which  is 
about  half  the  normal  size  and  hardly  more  than  skin  and  bones, 
but  is  as  deeply  pigmented  as  any  of  the  larger  specimens. 

Tornier's  experiments  with  yolk  removal  were  also  imitated 
with  the  Hyla  and  Eana  tadpoles.  Here  again  it  was  found  that 
the  tadpoles  which  had  lost  yolk,  though  smaller  than  the  others, 


62  University  of  California  Publications  in  Zoology    (TOL-  n 

were  not  necessarily  lighter  colored.  A  part  of  the  yolk  was 
removed  from  a  number  of  tadpoles  by  sucking  it  through  a 
capillary  tube.  Approximately  half  of  the  yolk  was  removed 
from  the  Eana  tadpoles  and  a  larger  proportion  from  the  Hyla 
tadpoles.  The  yolk  was  not  removed  until  the  embryos  were 
quite  large — nearly  ready  to  hatch — so  it  is  probable  that  in  the 
case  of  Hyla  at  least,  a  portion  of  the  alimentary  tract  was 
removed  with  the  yolk. 

The  two  lots  of  Eana  tadpoles,  those  from  which  the  yolk  had 
been  removed,  and  the  control  presented  practically  the  same 
appearance:  both  were  dark  grey,  but  those  which  had  suffered 
the  loss  of  yolk  were  no  lighter  than  the  control  (table  2). 

TABLE  2 

Length  measurements  of  tadpoles  from  which   yolk  has  been  removed. 
Measurements  represent  average  lengths  of  all  tadpoles  in  the  dish. 

A.  Eana  tadpoles.     Yolk  removed  Feb.  2,  1912 

Feb.  26 

Length  measurements  in  mm.  taken  f A ^ 

f A ^    Number  of 

Feb.  6        Feb.  9       Feb.  13      Feb.  20      Feb.  26       tadpoles     Color 

Yolk  removed         10.3         12.5         13.8         15.0         14.8         8         Medium 
Control  9.9         12.2         14.5         14.9         14.9         5         Medium 

B.  Hyla  tadpoles.    Yolk  removed  Feb.  19 

Mar.  2 

Length  measurements  in  mm.  taken       f A > 

f A ^  Number  of 

Feb.  20     Feb.  22     Feb.  26     Mar.  2         Color  tadpoles 

Yolk  removed  7.87  9.08  9.55  9.4  A  few  somewhat  3 

( 10  tadpoles )  lighter  than  control 

Control  8.1  9.51  10.15  10.05  Medium  9 

(10  tadpoles) 

The  two  lots  of  Hyla  tadpoles  showed  slight  differences.  Some 
of  the  individuals  that  had  suffered  the  loss  of  yolk  were  as  dark 
as  the  control  tadpoles  but  others  were  a  little  lighter.  Micro- 
scopical examination  shows  that  these  lighter  individuals  have 
about  the  same  number  of  pigment  spots,  but  that  some  of  the 
spots  are  smaller  and  some  are  a  little  lighter  than  those  of 
the  control  tadpoles. 

This  difference  seems  to  me  to  be  a  direct  result  of  the  loss 
of  nourishment — the  amount  of  pigment  is  less  just  as  the  length 
and  width  measurements  are  less  and  the  muscles  and  other 


19131     Johnson:  Pigment  Formation  in  Amphibian  Larvae         63 

tissues  appear  more  transparent.  The  yolk  loss  has  meant  to 
the  tadpole  a  loss  of  substances  from  which  directly  or  indirectly 
pigment  is  ultimately  formed,  as  well  as  a  loss  of  tissue  forming 
substance.  The  very  smallness  of  the  differences  indicates  that 
they  are  due  to  a  lack  of  material  for  tissues  and  pigment  build- 
ing rather  than  to  a  using  up  of  pigment  once  formed. 

This  view  is  supported  by  Riddle's  investigation  of  pigment 
formation  in  the  feathers  of  young  birds.  Although  there  is 
little  definite  knowledge  concerning  the  formation  of  the  par- 
ticular melanins  present  in  this  instance  or  the  other  cases  cited, 
it  is  highly  probable  that  they  are  formed  similarly  and  that 
they  are  equally  dependent  upon  or  independent  of  nutrition. 

After  carrying  on  feeding  experiments  with  young  birds, 
Riddle  (1908)  concludes  that  pigment  and  barbule  forming  cells 
are  both  reduced  in  rate  of  production  relative  to  growth  in 
certain  other  parts  of  the  feather  because  of  the  less  favorable 
relations  which  the  pigment  producing  cells  bear  to  the  nutriment 
carried  by  the  blood.  He  says,  "In  just  the  same  way  that  a 
lack  of  nutrition  checks  the  production  of  barbule  forming  cells, 
it  reduces  the  amount  of  pigment  formed  and  taken  up  by  the 
barbule  cells." 

There  is  in  this  instance  as  in  the  case  of  the  tadpole,  a  loss 
of  certain  tissue  building  substances  along  with  a  loss  of  pigment 
forming  substances.  The  "less  favorable  relations  which  the 
pigment  producing  cells  bear  to  the  nutriment  carried  by  the 
blood"  may  make  the  difference  between  daily  and  nightly 
growth  more  marked  here  than  in  other  places  but  the  difference 
is  probably  present  in  other  parts  though  from  the  nature  of  the 
structure  it  is  less  easily  discovered. 

Similarly,  in  reporting  observations  on  Amblystoma  tigrinum, 
Powers  (1908,  p.  38)  says:  "I  have  kept  many  adults,  young  and 
old,  for  three  and  even  four  years,  and  have  subjected  them  to 
very  varying  conditions  of  nutrition,  temperature,  etc.,  and  by 
means  of  photographs  I  have  compared  the  appearance  of  many 
during  successive  seasons.  Starvation  does,  of  course,  produce 
a  marked  effect  on  many  organs  and  upon  the  animal's  whole 
appearance,  save  color." 


64  University  of  California  Publications  in  Zoology    [VOL.  11 

These  instances  and  the  results  of  the  experiments  all  point 
to  the  same  conclusion,  that  with  reduced  nutrition  there  is  a 
reduced  pigment  production  along  with  a  reduction  of  tissue  or 
tissue  formation  with  the  result  in  the  case  of  the  tadpole  that 
the  color  of  the  animal  appears  neither  darker  nor  lighter. 

D.  EFFECT  OF  VAEIOUS  KINDS  OF  FOOD  UPON  PIGMENTATION 

Of  especial  interest  in  a  discussion  of  the  effect  of  food  upon 
the  color  of  animals  are  the  following  classical  examples  cited  by 
Darwin  (1868,  vol.  2,  p.  337)  in  "The  Variation  of  Animals  and 
Plants  under  Domestication."  He  says:  "It  is  well  known  that 
hemp-seed  causes  bullfinches  and  certain  other  birds  to  become 
black.  Mr.  Wallace  has  communicated  to  me  some  much  more 
remarkable  facts  of  the  same  nature.  The  natives  of  the  Amaz- 
onian region  feed  the  common  green  parrot  (Chrysotis  f estiva, 
Linn.)  with  the  fat  of  large  Siluroid  fishes,  and  the  birds  thus 
treated  become  beautifully  variegated  with  red  and  yellow 
feathers.  In  the  Malayan  archipelago,  the  natives  of  Gilolo  alter 
in  an  analogous  manner  the  colours  of  another  parrot,  namely  the 
Lorius  garrulus  Linn.,  and  thus  produce  the  Lori  rajah  or  King- 
Lory.  These  parrots  in  the  Malay  Islands  and  South  America, 
when  fed  by  the  natives  on  natural  vegetable  food,  such  as  rice 
and  plantains,  retain  their  proper  colours.  Mr.  Wallace  has,  also, 
recorded  (A.  R.  Wallace,  Travels  on  the  Amazon  and  Rio  Negro, 
p.  294)  a  still  more  singular  fact.  'The  Indians  (of  S.  America) 
have  a  curious  art  by  which  they  change  the  colours  of  the 
feathers  of  many  birds.  They  pluck  out  those  from  the  part  they 
wish  to  paint,  and  inoculate  the  fresh  wound  with  the  milky 
secretion  from  the  skin  of  a  small  toad.  The  feathers  grow  of  a 
brilliant  yellow  colour,  and  on  being  plucked  out,  it  is  said,  grow 
again  of  the  same  colour  without  any  fresh  operation. ' ' 

Romanes  (1895,  vol.  2,  p.  218)  cites  the  above  instances  of 
changes  in  the  plumage  of  birds  and  the  fact  also  noted  by 
Darwin  that  canaries  become  red  when  fed  on  cayenne  pepper, 
and  adds:  "Dr.  Sauermann  has  recently  investigated  the  subject 
experimentally;  and  finds  that  not  only  the  finches,  but  likewise 
other  birds,  such  as  fowls,  and  pigeons,  are  subject  to  similar 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         65 

variations  of  colour  when  fed  on  cayenne  pepper;  but  in  all 
cases  the  effect  is  produced  only  if  the  pepper  is  given  to  the 
young  birds  before  their  first  moult.  Moreover,  he  finds  that  a 
moist  atmosphere  facilitates  the  change  of  colour,  and  that  the 
ruddy  hue  is  discharged  under  the  influence  either  of  sunlight 
or  of  cold.  Lastly,  he  has  observed  that  sundry  other  materials 
such  as  glycerine  and  aniline  dyes,  produce  the  same  results ;  so 
there  can  be  no  doubt  that  organic  compounds  probably  occur 
in  nature  which  are  capable  of  directly  affecting  the  colours  of 
plumage  when  eaten  by  birds." 

These  facts  indicate  that  significant  variations  in  color  are 
produced  by  different  kinds  of  food  and  the  following  experi- 
ments with  tadpoles  bear  out  the  suggestion. 

Both  Rana  and  Hyla  tadpoles  were  used  for  these  experi- 
ments. Different  bunches  of  eggs  were  separated  so  that  the 
larvae  used  for  one  set  of  experiments  were  from  a  single  egg 
mass.  As  soon  as  the  tadpoles  hatched  they  were  divided  into 
different  groups  and  each  group  was  fed  on  one  kind  of  food. 
The  water  in  the  dishes  was  changed  every  other  day  and  the 
tadpoles  given  fresh  food.  In  the  later  experiments  the  table  and 
the  sides  of  the  dishes  were  covered  with  black  paper.  This 
allowed  the  light  to  enter  only  from  above  and  insured  equal 
illumination  for  all  the  dishes.  The  foods  first  used  were  beef 
liver,  egg  yolk,  egg  albumen,  beef  suet,  brown  and  white  beans, 
white  bread,  and  fish.  The  foods  were  all  cooked  by  boiling  and 
finely  divided  by  being  put  through  a  sieve  or  rubbed  between 
the  fingers.  A  few  measurements  given  in  the  following  tables 
will  show  the  size  relations  fairly  well.  The  intensity  of  the 
color  is  also  noted  (table  3). 


66 


University  of  California  Publications  in  Zoology    [VOL.  11 


TABLE  3 

Length  measurements  and  color  of  tadpoles  fed  on  different  kinds  of  food. 
Measurements  represent  average  of  several  typical  tadpoles  from  each 
dish. 


Food 
Liver 
Yolk 
Bread 
Fish 
-Albumen 


A.  Eyla.    Experiment  started  Feb.  18,  1911 


Length  in  mm. 

April  18 
38.0  mm. 
40.0 
31.0 
22.5 
26.0 


Color 
Dark 
Light 
Medium 
Medium 
Dark 


Number  of  tadpoles 
Apr.  8 


Feb.  18 

7 
7 
7 
7 
7 


B.  Hyla.    Experiment  started  Apr.  17,  1911 


Length  in  mm. 


Number  of  tadpoles 


Food 
Liver 

May  8 
25 

June  3 
37 

Color 
Dark 

White  beans 

22 

35 

Medium 

Brown  beans 

21 

40 

Medium 

Suet 

14 

16 

Dark 

Apr.  17 
4 
5 
5 
5 


June  3 
3 
4 
5 
4 


C.  Hyla.    Experiment  started  Apr.  17,  1911 

Length  in  mm.  Number  of  tadpoles 


Food 
Egg 

White  beans 
Brown  beans 
Suet 


Apr.  22 
13 
13 
11 
11 


May8 
22 
20 
22 
14 


Color 
Light 
Medium 
Medium 
Dark 


To  see  if  it  would  be  possible  to  lighten  the  color  of  tadpoles 
that  were  already  strongly  pigmented,  some  very  dark  tadpoles 
collected  from  a  pond  were  fed  upon  yolk  of  egg.  In  nine  days 
they  were  distinctly  lighter  than  tadpoles  from  the  same  source 
that  had  been  kept  in  the  aquarium  on  a  mixed  diet.  I  do  not 
regard  this  as  a  disappearance  of  pigment  already  present  in 
the  tadpole,  but  rather  as  a  decrease  in  pigment  production 
relative  to  growth.  The  proportion  of  pigment  formed  is  less 
with  egg  yolk  as  food,  so  that  the  ratio  of  pigment  to  body  size 
decreases,  until  within  nine  days,  the  difference  in  color  is 
noticeable. 

If  we  assume  an  oxidase  reaction  as  the  basis  of  pigment 
formation  in  the  tadpoles  we  explain  a  decrease  in  the  rate  of 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         67 

pigment  formation  by  a  decrease  in  the  amount  or  power  of  the 
oxidase  or  by  a  decrease  in  the  amount  of  the  chromogen  or  by 
the  presence  of  something  which  inhibits  the  action  of  the  oxidase. 

The  oxidase  in  these  experiments  was  certainly  not  admin- 
istered to  the  animal  directly,  since  all  of  the  foods  were  boiled 
before  being  given,  and  oxidases  are  destroyed  at  this  tempera- 
ture. Therefore  the  oxidase  was  produced  by  the  organism.  If 
the  difference  in  pigmentation  was  due  to  a  greater  amount  of 
oxidase,  the  later  could  have  been  furnished  only  indirectly  by 
the  foods. 

In  considering  the  amount  of  chromogen  contributed  by  the 
foods,  one  must  consider  their  proteid  content,  since  tyrosin,  a 
common  chromogen,  is  a  decomposition  product  of  the  digested 
proteids.  A  study  of  the  analyses  of  the  various  foods  shows  that 
the  pigmentation  is  not  correlated  with  the  amount  of  proteid, 
since  egg  yolk  which  produced  little  pigment  contains  a  large 
percentage  of  proteid,  larger  than  beans,  which  produced  more 
pigment.  From  the  table  it  will  be  seen  that  while  pigmentation 
may  depend  upon  the  kind  of  proteid  fed,  it  cannot  depend 
upon  the  gross  amount  of  proteid  (table  4).  A  few  tabulations 
of  the  cleavage  products  of  these  foods  are  available  (see  table 
10).  It  is  notable  that  in  these  ov-albumen  shows  a  larger  per- 
centage (2.4%)  of  tyrosin  than  does  vitellin  (1.6%).  The 
differences  are  very  slight,  but  it  will  be  remembered  that  the 
differences  in  pigmentation  are  also  small,  and  that  tyrosin  is  so 
slightly  soluble  in  water  that  artificial  melanins  are  formed  with 
very  dilute  solutions  of  the  chromogen. 

TABLE  4 

Analyses  of  the  foods  used  in  experiments,  from  Hammarsten,  ' '  A  Text 
Book  of  Physiological  Chemistry,"  trans,  by  Mandel  (1911,  p.882-3). 

1000  parts  contain  Relation  of  1:2:3 


123456  1:2:3 

Proteids 

and  ex-  Carbo- 

tractives  Fat  hydrates  Ash  Water  Waste 

....  100  28  0 

....  100  192  0 

....  100  7  7 

12  100  4  244 


Beef  liver 

196 

56 

11 

17 

720 

Egg  yolk 

160 

307 

.... 

13 

520 

Egg  albumen 

103 

7 

7 

8 

875 

Beans 

27 

1 

66 

6 

888 

68  University  of  California  Publications  in  Zoology    [VOL.  11 

It  is  quite  possible  that  the  two  causes,  perhaps  three,  always 
operate  to  some  degree,  that  a  strong-pigment  forming  food  such 
as  liver  may  furnish  in  the  digestive  process  a  large  proportion 
of  tyrosin  or  some  other  chromogen,  and  at  the  same  time  fur- 
nish few  inhibiting  factors.  The  third  factor,  not  so  easily 
accessible  to  experiment,  may  be  an  increased  formation  of  the 
oxidase  by  a  strong  pigment-forming  food. 

The  large  proportion  of  fat  in  egg  yolk  suggested  that  the 
lecithin  of  the  yolk  might  be  the  element  that  in  some  way 
brought  about  a  decrease  in  pigment  production.  The  results  of 
Danilewsky  supported  the  suggestion  that  lecithin  might  be  an 
inhibitor,  possibly  the  only  one. 

Danilewsky  (1805)  placed  tadpoles  in  a  solution  containing 
one  part  of  lecithin  to  15,000  parts  of  water.  Other  tadpoles  were 
kept  in  water  alone  to  serve  as  a  control.  The  former  became 
three  times  heavier  and  nearly  twice  as  long  as  the  corresponding 
controls.  He  thought  the  effect  was  due  to  the  stimulating  effect 
of  the  lecithin  rather  than  to  any  great  amount  of  nourishment 
contained  in  it.  He  adds :  * i  II  f aut  encore  noter  que  tous  les 
tetards  lecithiniques  etaient  beaucoup  moins  pigmentes  que  les 
larves  de  controle. "  He  found  also  that  when  lecithin  is  injected 
into  young  rabbits  or  dogs  there  is  a  marked  increase  in  their 
growth. 

Miss  King  (1907)  conducted  a  series  of  feeding  experiments 
with  large  numbers  of  toad  tadpoles.  She  refers  to  an  apparent 
stimulating  effect  of  lecithin  in  egg  yolk  and  mentions  meat  as  a 
good  pigment  producer. 

Goldfarb  (1910,  p.  272)  reviews  the  results  of  these  and  other 
experiments  with  lecithin  and  reports  upon  similar  investigations 
of  his  own.  He  says,  ''Frog  and  toad  tadpoles  were  placed  in 
graded  lecithin  solutions  ranging  from  one  part  of  lecithin  in 
20,000  of  water  to  toxic  solutions  of  %0  and  kept  therein 
throughout  their  period  of  metamorphosis  (33  to  51  days) .  Other 
conditions,  such  as  temperature,  amount  of  solution,  number  of 
tadpoles  in  each  dish,  food,  etc.,  were  constant  for  each  series. 
The  control  tadpoles  showed  at  the  end  of  the  experiment  a 
variation  of  9  to  53  per  cent  in  weight,  3  to  44  per  cent  in  length. 
About  1,000  tadpoles  kept  in  lecithin  solutions  showed  a  maxi- 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         69 

mum  variation  of  23  to  64  per  cent  for  the  same  period  of  time. 
There  was  no  definite  increase  in  size  or  weight  in  the  increasing 
concentrations  of  lecithin,  nor  was  there  a  definite"  gain  among 
those  in  lecithin  taken  as  a  whole  as  contrasted  with  the  controls. 

These  facts  led  to  farther  investigation  of  lecithin,  its  effect 
both  upon  the  tyrosinase  reaction  and  pigmentation  in  the  larvae. 


E.  EFFECT  OF  LECITHIN  AND  VABIOUS  FOODS  USED  IN  EXPERI- 
MENTATION,  UPON  THE  TYROSINASE  REACTION 

The  following  tests  in  vitro  of  the  effect  of  the  various  foods 
upon  the  tyrosinase  reaction  support  the  suggestion  that  the 
lecithin  in  egg  yolk  inhibits  pigment  formation. 

Tyrosinase  for  the  tests  was  obtained  from  different  sources 
as  follows: 

(1)  Fly  larvae  from  six  to  ten  days  old  were  washed,  chloro- 
formed, and  then  ground  with  distilled  water  in  a  glass  mortar. 
The  aqueous  extract  thus  obtained  was  filtered  through  glass  or 
cotton  wool,   and  used  immediately.     This  solution  is  a  very 
powerful  one  and  turns  orange  pink  throughout  before  one  can 
get  it  measured  into  the  test  tubes. 

(2)  Meal  worms  were  extracted  in  the  same  way  and  the 
course  of  the  reaction  is  much  the  same,  but  the  solution  does  not 
color  quite  as  rapidly  as  in  the  case  of  the  fly  larvae. 

(3)  Mushrooms  were   extracted  with  glycerine   and  water, 
after  having  been  ground  in  a  glass  mortar.     The  extract  was 
filtered  through  ordinary  filter  papers. 

(4)  Potatoes  were  scraped  and  extracted  with  water  for  about 
an  hour.    This  extract  was  filtered  through  filter  papers  and  used 
as  soon  as  possible.     The  solution  is  a  pale  orange  pink  by  the 
time  it  is  measured  out  into  the  test  tubes. 

A  saturated  solution  of  tyrosin  in  water  was  usually  added 
to  these  solutions  for  the  experiments,  but  the  extracts  already 
contained  a  chromogen  since  all  of  them  darken  after  standing 
for  half  an  hour.  Since  solutions  color  up  in  the  same  way  when 
tyrosin  is  added  as  when  it  is  not  added,  it  seems  likely  that 
tyrosin  is  the  chromogen  already  present  in  the  solution.  The 
extract  from  fly  larvae  gives  a  positive  result  for  tyrosin  when 


70  University  of  California  Publications  in  Zoology    [VOL.  11 

tested  with  Million's  reagent.  Owing  to  the  fact  that  the  tyro- 
sinase  when  isolated  by  precipitation  with  alcohol  or  other  agents, 
is  much  less  powerful  than  the  original  extract,  in  most  of  these 
experiments  no  attempt  was  made  to  isolate  the  oxidase.  Kastle 
(1910,  p.  63)  has  experienced  the  same  difficulty  and  says, 
"Inasmuch  as  we  have  no  criterion  for  judging  of  the  absolute 
purity  of  a  ferment,  it  is  very  doubtful  whether  much  is  gained 
by  the  attempt  to  isolate  laccase  and  the  other  oxidases  in  pure 
condition. ' ' 

Two  series  of  experiments  were  made.  In  the  first,  the  various 
foods  under  investigation  were  cooked  and  ground  and  then 
added  to  solutions  of  tyrosin  and  tyrosinase;  in  the  second  the 
foods  were  digested  in  pancreatin  and  pepsin  and  the  filtrates 
used. 

SERIES  A.  UNDIGESTED  FOODS 

For  these  experiments  0.2  gm.  of  the  food  was  weighed  out 
after  it  had  been  thoroughly  cooked  by  boiling.  This  portion  was 
ground  in  a  glass  mortar  with  10  cc.  of  distilled  water  and  added 
to  the  tyrosinase  solution  with  or  without  tyrosin.  The  results 
are  more  or  less  variable,  but  certain  facts  may  be  stated  with 
assurance  to  hold  for  all  sorts  of  tyrosinase  used. 

Albumen  inhibits  the  action  of  the  tyrosinase  the  least  of 
any  of  the  foods.  Yolk  and  liver  inhibit  the  reaction  more  than 
albumen — liver  usually  inhibiting  more  than  yolk.  Liver  allowed 
to  spoil  and  then  cooked  inhibits  the  reaction  completely,  giving 
a  white  precipitate  and  a  clear,  colorless  liquid.  Liver  that  has 
been  kept  for  several  days,  but  cooked  from  time  to  time  to 
prevent  its  putrefaction,  inhibits  the  reaction  less  than  does  the 
fresh  liver.  Fresh  and  stale  eggs  inhibit  the  reaction  equally. 
Lecithin*  inhibits  the  action  of  the  tyrosinase  markedly — the 
color  appearing  slowly  and  only  toward  the  surface  of  the  liquid. 


*  Lecithin  was  prepared  as  follows :  Yolks  of  hen 's  eggs  were  washed  in 
water  without  breaking  the  membrane.  An  equal  volume  of  10  per  cent 
sodium  chloride  solution  was  added  and  the  mixture  extracted  in  a  separ- 
atory  funnel  with  two  volumes  of  ether.  To  the  ether  fraction  was  added 
an  equal  volume  of  acetone.  The  precipitated  lecithin  was  dried  in  a 
sulphuric  acid  dessicator. 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         71 

Pepsin  and  pancreatin  (Merck  in  both  cases)  were  tested  in 
the  same  way,  0.2  grams  being  ground  in  10  cubic  centimeters  of 
water  and  the  tyrosinase  solution  added.  Pepsin  inhibits  the 
reaction  markedly,  sometimes  entirely.  The  pancreatin  tubes,  on 
the  other  hand,  are  usually  as  dark  as  the  control. 

Although,  in  these  tests,  liver  inhibits  the  reaction  more  than 
egg  yolk,  the  result  is  reversed  when  the  foods  are  digested, 
digestion  apparently  lessening  the  inhibitory  action  of  the  liver. 
As  one  would  expect,  whatever  effects  the  foods  may  have  on  the 
reaction  before  digestion,  these  effects  are  more  or  less  changed 
after  digestion. 

SERIES  B.  DIGESTED  FOODS 

In  these  experiments  the  foods  were  digested  with  pepsin  and 
pancreatin  and  the  nitrates  added  to  the  tyrosinase  solution.  In 
each  case  five  grams  of  the  food  were  ground  in  a  glass  mortar 
with  ten  cubic  centimeters  of  distilled  water,  and  digested  with 
0.6  gram  of  pepsin  or  pancreatin.  A  few  drops  of  toluol  were 
added  and  the  tubes  were  kept  in  a  warm  place.  After  digesting 
for  forty-eight  hours,  the  masses  were  filtered.  The  tyrosin  and 
tyrosinase  were  mixed  and  the  solution  measured  out  from  a 
burette  into  test  tubes  containing  a  few  drops  of  toluol  and 
seven  drops  of  filtrate  or,  in  the  case  of  the  control,  seven  drops 
of  distilled  water. 

By  the  time  the  solutions  have  been  introduced  into  the  tubes 
containing  the  filtrates  and  toluol  and  the  tubes  arranged  for 
inspection  they  are  all  a  delicate  orange  pink.  The  yolk  tubes 
soon  become  paler,  passing  into  a  pinkish  white  and  the  pan- 
creatins  soon  lose  the  pink  tinge  entirely,  passing  into  a  light 
greenish  brown,  at  the  same  time  turning  a  smoky  black  at  the 
surface.  This  black  color  gradually  deepens  until  the  solution  is 
jet  black  throughout.  If  after  the  reaction  has  gone  on  for 
twelve  hours,  the  tubes  are  shaken,  the  three  are  all  very  dark, 
but  the  yolk  tube  is  brown  or  greenish  brown  while  the  liver  and 
albumen  tubes  are  jet  black. 

The  pepsin  tubes  go  through  the  reaction  more  slowly,  show- 
ing much  of  the  orange  pink  color  after  the  pancreatin  tubes 
have  lost  their  pink  color  and  have  become  black  at  the  surface. 


72  University  of  California  Publications  in  Zoology    ITOL-  n 

The  darkening  toward  the  surface  proceeds  more  slowly  also 
showing  more  of  the  red  and  mahogany  tints  before  reaching 
black.  The  solutions  become  as  dark  as  the  others  if  left  long 
enough,  and  when  shaken  show  the  same  slight  difference  between 
the  yolk  and  the  liver  and  albumen.  Liver  and  albumen  tubes 
are  often  the  same  shade,  but  if  there  is  a  difference,  the  albumen 
tube  is  usually  darker.  The  control  tube  acts  more  like  pepsin 
than  pancreatin,  retaining  its  pink  color  for  some  time  as  the 
pepsin  filtrates  do. 

The  more  rapid  formation  of  color  in  the  pancreatin  tubes 
as  compared  with  the  pepsin  or  control  tubes  is,  I  think,  due  in 
part  to  the  fact  that  the  filtrates  contain  products  of  digestion, 
among  which  are  probably  some  which  serve  as  chromogens. 
Tyrosin  is  present  among  the  cleavage  products  of  most  foods; 
and  there  may  be  other  chromogens  present.  Their  presence 
would  account  for  the  fact  that  the  test  tubes  containing  pan- 
creatin filtrates  color  more  quickly  than  the  control  tubes  do. 
The  tubes  containing  pepsin  filtrates,  on  the  other  hand,  prob- 
ably contain  enough  pepsin  to  slow  down  the  reaction,  since  we 
found  that  pepsin  alone  inhibits  the  reaction  markedly.  It  is 
also  possible  that  they  contain  less  chromogen  than  the  pancreatin 
filtrates.  Probably  both  factors  operate  to  produce  the  result,  a 
color  reaction  which  generally  runs  parallel  with  that  of  the 
control  tubes. 

Gessard  (1901)  obtained  a  similar  result  when  he  found  that 
forty  drops  of  blood  serum  from  a  calf  retarded  the  tyrosinase 
reaction  nine  days,  while  fifty  drops  of  water  retarded  it  for  a 
month.  It  is  highly  probable  that  some  chromogen  was  present 
in  the  blood  serum  which  would  at  least  contribute  more  toward 
pigment  formation  than  would  any  elements  contained  in  an 
equal  amount  of  water. 

Though  the  results  of  the  experiments  are  not  altogether  con- 
stant, the  variability  in  the  reaction  is  probably  to  be  accounted 
for  partly  by  the  fact  that  different  eggs  and  livers  were  used 
in  different  experiments,  partly  by  the  fact  that  the  foods  may 
have  reached  different  stages  of  digestion  in  the  different  sets  of 
experiments,  and  perhaps  by  variability  of  other  and  unknown 
factors.  In  spite  of  these  differences,  however,  the  experiments 


1913J     Johnson:  Pigment  Formation  in  Amphibian  Larvae         73 

show  plainly  that  lecithin  and  egg  yolk  inhibit  to  some  extent 
the  action  of  certain  tyrosinases  of  vegetable  and  animal  origin. 
This  being  so,  what  will  be  the  effect  when  lecithin  is  fed  to  the 
tadpoles  along  with  a  food  which  produces  marked  pigmentation  ? 

F.  EFFECT  OF  LECITHIN  UPON  PIGMENTATION 

Experiments  were  accordingly  made  to  determine  the  effect 
of  lecithin  when  it  is  fed  along  with  a  food  that  ordinarily  pro- 
duces considerable  pigment.  When  lecithin  was  mixed  with  the 
food  it  was  placed  in  the  dishes  every  other  day  after  the  dishes 
had  been  cleaned  and  the  food  and  water  renewed.  The  amount 
of  lecithin  used  was  not  weighed  each  time,  but  it  probably 
varied  between  fifteen  and  twenty  milligrams  in  weight.  It 
dissolved  after  eight  or  ten  hours. 

Of  two  lots  of  Rana  tadpoles,  one  was  fed  albumen  and  the 
other  albumen  and  lecithin.  Following  are  the  notes  on  the 
experiments.  Table  5  shows  the  length  measurements  on  different 
dates. 

TABLE  5 

Length  measurements  of  Sana  tadpoles  fed  on  albumen  and  albumen  plus 
lecithin.  Length  measurements  represent  average  length  of  all  the 
tadpoles.  Eight  tadpoles  in  each  lot. 

Eana.     Experiment  started  Feb.  6,  1912 
Length  in  mm. 


Food  Feb.  19          Feb.  26  Mar.  5          Mar.  25 

Albumen  17.6  18.6  19.3  21.5 

Albumen  +  lecithin  18.2  21.1  23.0  26.5 

Feb.  21.  Tadpoles  fed  on  albumen  plus  lecithin  are  lighter  than  those 
fed  on  albumen  alone.  The  difference  is  small  but  distinct. 

Mar.  5.  Tadpoles  fed  on  albumen  plus  lecithin  are  very  plainly  lighter 
and  larger  than  tadpoles  fed  on  albumen. 

Mar.  14.     (Same  as  Mar.  5.) 

Mar.  25.  Color  difference  seen  before  is  now  scarcely  noticeable.  Size 
difference  very  plain. 

Apr.     2.     Color  difference  plain  once  more. 

Apr.  10.  On  account  of  the  size  difference  it  is  difficult  to  compare  the 
two  sets  of  tadpoles  with  accuracy.  One  tadpole  fed  on  albu- 
men plus  lecithin  is  darker  than  the  others  but  the  rest  are 
lighter  than  the  albumen-fed  tadpoles,  showing  a  yellowish 
brown  tinge  rather  than  black. 


74  University  of  California  Publications  in  Zoology    [VOL-  n 

The  same  experiment  was  carried  out  with  Hyla  tadpoles  with 
the  same  results  (table  6). 


TABLE  6 

Length  measurements  of  Hyla  tadpoles  fed  on  albumen  and  albumen  plus 
lecithin.  Length  measurements  represent  average  length  of  all  the 
tadpoles  in  the  dish. 

Hyla.    Experiment  started  Feb.  21 
Length  in  mm. 


Food  Feb.  26          Mar.  5          Mar.  25 

Albumen  12.5  17.1  22.0 

Albumen  +  lecithin        12.3  16.9  25.6 

Mar.  5.  Tadpoles  fed  on  lecithin  plus  albumen  are  distinctly  lighter  than 
the  others.  The  tadpoles  fed  on  albumen  alone  have  slender, 
pinched  bodies,  while  the  others  are  the  normal  shape  and 
size. 

Mar.  25.     The  color  difference  is  scarcely  noticeable. 

Apr.  10.  Some  tadpoles  on  lecithin  plus  albumen  are  as  dark  as  those  on 
albumen  alone,  but  most  of  them  are  lighter. 


Liver  plus  lecithin,  and  liver  alone,  were  fed  to  two  different 
sets  of  Rana  tadpoles  with  the  following  results  (table  7)  : 

TABLE  7 

Length  measurements  of  Eana  tadpoles  fed  on  liver  and  liver  plus  lecithin. 
Length  measurements  represent  average  length  of  all  the  tadpoles. 
Eight  tadpoles  in  each  lot. 

Eana.    Experiment  started  Feb.  6,  1912 
Length  in  mm. 


Food  Feb.  20          Feb.  26  Mar.  6  Mar.  14 

Liver  21.4  24.2  27.4  30.25 

Liver  +  lecithin  20.6  24.05  28.2  30.29 

Feb.  21.     Tadpoles  fed  on  liver  alone  are  darker  than  the  others. 
Feb.  26.     Tadpoles  fed  on  liver  alone  are  darker  than  the  others. 
Mar.  14.     Some  about  the  same  color,  others  darker  in  dish  containing 
liver  alone. 

The  same  experiments  with  Hyla  gave  the  following  results 
(table  8)  : 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         75 


TABLE  8 

Length  measurements  of  Hyla  tadpoles  fed  on  liver  and  liver  plus  lecithin. 
Length  measurement  represents  average  length  of  all  the  tadpoles. 
Six  tadpoles  in  each  lot. 

Eyla.     1912 

Length  in  mm. 


Food  Mar.  5  Mar.  14 

Liver  19.5  24.4 

Liver  +  lecithin  19.6  26.2 

Mar.  14.     Tadpoles  fed  on  liver  plus  lecithin  are  lightest — have  a  greenish 

tinge  while  others  are  black. 
Mar.  25.     Tadpoles  fed  on  liver  are  all  dead. 

Two  sets  of  Hyla  tadpoles  were  fed  on  egg  yolk  and  egg  yolk 
plus  lecithin  (table  9)  : 

TABLE  9 

Length  measurements  of  tadpoles  fed  on  yolk  and  yolk  plus  lecithin. 
Length  measurements  represent  average  length  of  all  the  tadpoles. 
Eight  tadpoles  in  each  lot. 

Hyla.     1912 

Length  in  mm. 

Food  Mar.  5  Mar.  14          Mar.  25 

Yolk  15.7  18.2  22.6 

Yolk  +  lecithin  15.6  17.6  22.1 

Mar.  14.     Sizes  are  uneven.    No  marked  color  differences.     A  few  lighter 
individuals  among  the  tadpoles  fed  on  yolk. 

Apr.     1.     Tadpoles  fed  on  yolk  average  slightly  darker  than  the  others — 

pigmentation  very  uneven. 
(Tadpoles  accidentally  mixed,  experiment  discontinued.) 


The  experiments  plainly  show  that  lecithin  lightens  the  color 
of  the  tadpoles.  It  is  not  clear  that  lecithin  plus  yolk  of  egg 
produces  lighter  colored  tadpoles  than  yolk  alone  does.  It  would 
appear  that  for  tadpoles  of  the  size  reached  by  these  at  least, 
additional  amounts  of  lecithin  do  not  lighten  the  tadpoles 
noticeably. 

Miss  King  (1907)  has  suggested  that  the  tadpoles  that  are 
fed  with  lecithin  are  not  so  thrifty  as  the  others.  Although  there 
has  been  a  slightly  larger  percentage  of  deaths  among  the  tad- 


76  University  of  California  Publications  in  Zoology    [VOL.  n 

poles  fed  on  yolk  of  egg  than  among  those  fed  on  liver,  the 
tadpoles  on  lecithin  mixed  with  other  foods  show  a  smaller  per- 
centage of  deaths  than  the  control  lots  receiving  no  lecithin. 

It  has  been  noticed  in  the  experiments  with  lecithin  in  con- 
nection with  other  foods,  that  the  color  differences  vary  from 
time  to  time.  Such  differences  have  been  noticed  in  all  the 
experiments  to  a  greater  or  less  extent.  In  general  the  differ- 
ence between  the  color  of  tadpoles  fed  on  yolk  and  those  fed  on 
liver  is  very  noticeable  at  the  end  of  the  first  week  and  continues 
to  be  very  marked  until  the  larvae  are  about  half  grown,  when  it 
becomes  less  distinct.  From  about  the  time  the  hind  limbs 
appear  until  the  metamorphosis  is  complete,  the  color  difference 
is  again  marked. 

These  differences  are  never  great  enough  to  suggest  an  actual 
disappearance  of  pigment  already  formed,  but  rather  a  slight 
difference  in  the  rate  of  pigment  production  at  different  stages 
of  development.  That  is,  the  pigment  production  in  tadpoles  fed 
on  egg  yolk  or  lecithin  though  at  all  times  less  than  in  the 
tadpoles  fed  on  liver  may  be  greater  at  one  time  than  at  another. 
It  is  possible  that  the  tadpole  at  different  stages  of  development 
is  differently  influenced  by  the  lecithin.  The  differences  may  be 
more  or  less  periodic,  but  more  observations  and  histological  data 
are  necessary  for  an  intelligent  discussion  of  this  phase  of  the 
problem. 

G.  EFFECT    OF   CERTAIN    OTHER    ORGANIC    SUBSTANCES   UPON 
THE  TYROSINASE  REACTION  AND  UPON  PIGMENTATION 

A  consideration  of  the  effect  of  various  other  chemicals  upon 
the  tyrosinase  reaction  is  important  if  we  grant  that  chemical 
substances  thus  profoundly  influence  pigment  formation. 

Abderhalden  and  Guggenheim  (1907)  found  that  n/100 
hydrochloric  acid  inhibits  the  action  of  tyrosinase  and  n/100 
sodium  hydroxide  retards  it  considerably.  Neutralization  of 
acid  or  alkali  fail  to  restore  the  original  activity  of  the  oxidase. 

Chodat  and  Staub  (1907)  observe  that  the  oxidation  of 
tyrosin  by  tyrosinase  is  diminished  by  glycin,  leucin,  and  alanin. 
They  find  that  tyrosin  acts  upon  certain  peptids  such  as  tyrosin 


19131     Johnson:  Pigment  Formation  in  Amphibian  Larvae         77 

anhydride  and  glycyl-ty rosin  anhydride,  giving  rise  to  yellow 
substances  which  do  not  become  black  as  does  tyrosin  itself. 
However,  a  mixture  a  glycyl-tyrosin  anhydride  with  glycin  gives 
with  tyrosinase  a  rose  color  changing  to  bluish  green,  with  alanin 
it  gives  a  deep  red,  and  with  leucin  a  deep  brown  color. 

Abderhalden  and  Guggenheim  (1907)  studied  the  action  of 
tyrosinase  from  Russula  delica  on  tyrosin  and  various  tyrosin- 
containing  polypeptids.  Aspartic  and  glutaminic  acids  and  other 
amino  acids  inhibited  the  action,  especially  if  they  were  present 
in  strong  solution.  Polypeptids  containing  tyrosin  residues  were 
colored  by  tyrosinase,  the  color  being  somewhat  modified  by  the 
nature  of  the  amino  acid  combined  with  the  tyrosin  in  the  poly- 
peptid.  They  conclude  that  the  character  of  the  pigment  result- 
ing from  the  action  of  tyrosinase  on  tyrosin  is  dependent  upon 
the  combination  in  which  the  tyrosin  exists. 


TABLE  10 

Cleavage  products  of  certain  animal  and  vegetable  proteids;  ov-albumin, 
vitellin,  and  gliadin  taken  from  Hammarsten  (1911),  gluten  from 
Plimmer  (1908). 


Glycocoll 

Alanine 

Valine 

Leucine 

Serine 

Aspartic  Acid 

Glutamic  Acid 

Cystin 

Phenylalanine 

Tyrosine 

Proline 

Oxyproline 

Tryptophane 

Histidine 

Arginine 

Lysine 

Ammonia 


Ov-albumin1       Vitellin4 


Gliadin 


a5 

&6 

0.0 

1.1 

0.02 

0.9 

2.1 

-f 

2.0 

2.66 

....  | 

2.4 

0.21 

0.33 

6.1  (  17 

11.0 

5.61 

6.00 

1.1 

.... 

0.13 

0.12 

1.5 

0.5 

0.5 

1.24 

8.0 

12.2 

37.33 

31.5 

0.33 



0.45 



4.4 

2.8 

2.35 

2.6 

2.4s 

1.6 

1.20 

2.37 

2.25 

3.3 

7.06 

2.4 

0.61 
3.16 
0.0 
5.11 


1.0 
1.7 
3.4 

0.0 


1  Abderhalden  and  Pregl  (1905). 

2  Levene  and  Beatty  (1907). 
SK.  Morner  (1901). 

4  Abderhalden  and  Hunter  (1906). 

5  Osborne  and  Clapp  (1906). 

8  Abderhalden  and  Samuely   (1905),  and  Abderhalden   (1909). 
7  Abderhalden  and  Malengreau ;  Kossel  and  Kutscher. 


Gluten,  from 

wheat7 

0.4 

0.3 


4.1 

0.7 
24.0 

1.0 
1.9 
4.0 


1.2 
4.4 
2.2 
2.5 


78  University  of  California  Publications  in  Zoology    LVoL-  n 

In  the  analyses  of  various  cleavage  products,  table  10,  gliadin 
and  gluten  are  notable  for  their  high  percentage  of  glutamic 
acid.  Glutamic  acid  is  noted  by  Abderhalden  and  Guggenheim 
as  an  inhibitor  of  the  tyrosinase  reaction  when  tyrosinase  from 
Russula  delica  is  used.  Accordingly  gliadin  and  gluten  (made 
from  white  flour,  dried,  ground,  and  fed  in  powdered  form)  were 
fed  to  different  sets  of  tadpoles.  These  foods  produce  so  little 
growth  that  the  results  are  very  inconclusive. 

The  results  with  liver,  yolk  and  gliadin  are  as  follows  (table 
11): 

TABLE  11 

Length  measurements  of  Hyla  tadpoles  fed  on  liver,  yolk,  and  gliadin. 
Length  measurements  represent  average  length  of  all  the  tadpoles  in 
a  dish. 

Hyla.     Experiment  started  Feb.  24,  1912 
Mar.  5  Mar.  25 


Food 
Liver 

Length 
in  mm. 

15.8 

Number  of 
tadpoles 

18 

Length 
in  mm. 

Number  of 
tadpoles 

0 

Yolk 

15.3 

18 

28.0 

14 

Gliadin 

12.5 

18 

15.0 

15 

Mar.  5.  Liver-fed  tadpoles  black.  Gliadin-fed  tadpoles  dark,  but  not 
so  dark  as  liver,  yolk-fed  tadpoles  lighter  with  a  few  very 
light  ones. 

Mar.  14.  Liver-fed  tadpoles  all  black;  yolk-fed  tadpoles — four  very  light, 
the  rest  medium;  gliadin,  one  light,  the  rest  medium. 

Of  three  sets  of  Rana  tadpoles  one  was  fed  on  raw  flour,  one 
on  gluten,  and  the  third  on  gliadin.  Here  again  the  growth  was 
so  slight  that  the  experiment  was  unsatisfactory  (table  12)  : 

TABLE  12 

Length  measurements  of  Eana  tadpoles  fed  on  flour,  gluten,  and  gliadin. 
Length  measurements  represent  the  average  length  of  all  the  tadpoles. 
Equal  number  of  tadpoles  at  the  beginning  of  the  experiment. 

Eana.     Experiment  started  Feb.  24,  1912 


Feed 

Length  in  mm. 

Number  of 
tadpoles 
May  6 

Mar.  6            Mar.  25^ 

Flour 

15.7                19.1 

9 

Gluten 

15.8             18.5 

1 

Gliadin 

15.7 

Mar.  25.     No  noticeable  color  difference. 

Apr.  10.     Only  one  gluten-fed  tadpole  left.    This  one  is  larger  and  slightly 
darker  than  the  average  of  the  flour-fed  tadpoles. 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         79 

The  same  experiment  with  Hyla  tadpoles  gave  the  following 
data  (table  13)  : 

TABLE  13 

Length  measurements  of  Hyla  tadpoles  fed  on  flour,  gluten,  and  gliadin. 
Length  measurements  represent  the  average  length  of  all  the  tadpoles 
in  a  dish. 

Hyla.  Experiment  started  Feb.  24,  1912 
Length  in  mm. 

Food  Mar.  6          Mar.  24 

Flour  13.0  18.3 

Gluten  13.3  19.6 

Gliadin  12.2  13.7 

Mar.  24.     Tadpoles  all  nearly  the  same  color,  —  if  any  difference  gluten- 
fed  are  darkest,  gliadin-fed  lightest. 

Gortner  (1911b)  finds  that  orcin,  resorcin,  and  phloroglucin 
inhibit  the  action  of  tyrosinase  extracted  from  potatoes,  meal 
worms,  and  the  periodical  cicada.  He  says  :  '  *  It  would  appear 
from  these  data,  that  aromatic  compounds  which  carry  two 
hydroxyl  groups  in  meta  position  to  each  other  may  act  as 
chemical  anti-oxidases  on  tyrosinase,  and  completely  inhibit  its 
action.  Other  oxidases  are  not  inhibited,  but  are  able  to  oxidize 
these  same  m-dihydroxl  compounds,  forming  colored  bodies  of  an 
unknown  nature." 

Phloroglucin  and  resorcin  were  mixed  with  liver  and  fed  to 
Hyla  tadpoles.  The  amounts  were  not  weighed,  but  about  fifteen 
milligrams  was  rubbed  up  with  the  liver  and  put  into  the  dish. 
The  tadpoles  on  resorcin  did  not  thrive,  most  of  them  died  very 
soon  (table  14)  : 

TABLE  14 

Length  measurements  of  tadpoles  fed  on  liver,  liver  -f-  resorcin,  and  liver 
+  phloroglucin.  Measurements  represent  the  average  length  of  all  the 
tadpoles  in  the  dish. 

Hyla.    Experiment  started  Mar.  18,  1912 

Mar.  25  Apr.  11 

Length 
Food  in  mm. 


Liver  14.1 

Liver  +  resorcin  15.4 

Liver  +  phloroglucin  14.3 


80  University  of  California  Publications  in  Zoology    1TOL-  n 

Mar.  25.  All  tadpoles  the  same  color. 
Apr.  1.  All  tadpoles  the  same  color. 
Apr.  11.  Of  the  four  remaining  tadpoles  on  liver  plus  phloroglucin,  two 

are  as  dark  as  the  six  tadpoles  fed  on  liver  alone,  and  two  are 

slightly  lighter. 

The  same  experiment  with  albumen  as  food  gives  the  follow- 
ing results  (table  15)  : 

TABLE  15 

Length  measurements  of  tadpoles  fed  on  albumen,  albumen  -f-  resorcin, 
and  albumen  +  phloroglucin.  Measurements  represent  the  average 
length  of  all  the  tadpoles  in  the  dish. 

Hyla.     Experiment  started  Mar.  18,  1912 

Length  in  mm. 
Food  Mar.  25 

Albumen  13.6 

Albumen  +  resorcin  13.1 

Albumen  +  phloroglucin  12.8 

Mar.  25.  Tadpoles  fed  on  albumen  plus  phloroglucin  are  very  slightly 
lighter  than  the  other  two  sets,  which  are  both  the  same  color. 

Apr.  11.  Tadpoles  fed  on  albumen  plus  phloroglucin  are  very  slightly 
lighter  than  those  on  albumen,  but  the  difference  is  scarcely 
noticeable. 

This  group  of  experiments,  as  will  be  noted,  was  tried  but 
once  and  with  a  small  number  of  tadpoles,  so  they  can  be  regarded 
only  as  a  beginning  in  this  direction.  The  results  indicate  that 
the  various  substances  may  affect  the  color  of  the  tadpoles  to 
some  extent. 


H.  HISTOLOGICAL  DIFFERENCES  BETWEEN  CHROMATOPHORES 
OF  LARVAE  FED  UPON  DIFFERENT  FOODS 

The  statements  as  to  the  color  differences  in  the  tadpoles  have 
been  made  thus  far  on  the  basis  of  their  appearance  to  the 
unaided  eye.  The  differences  as  shown  by  the  microscope  are  no 
less  marked  and  show  plainly  that  there  is  actually  less  pigment 
in  the  egg-fed  tadpoles  (plate  1). 

With  the  low  powers  of  the  microscope  the  black  epidermal 
chromatophores  of  the  dark  Kana  tadpoles  appear  to  be  greatly 
branched,  and  so  filled  with  pigment  that  they  come  very  close 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         81 

together,  while  in  the  light  egg-fed  tadpoles,  the  chromatophores, 
though  perhaps  not  less  numerous  are  more  slender  and  delicate 
and  very  little  branched.  In  very  light  specimens  the  pigment 
is  in  spots  that  are  scarcely  elongated  at  all  (pi.  1,  figs.  5-8). 

The  form  of  the  chromatophores  in  the  Hyla  is  somewhat 
different.  The  body  of  the  chromatophore  is  not  so  long,  but  the 
branching  processes  are  longer  and  finer  and  in  dark  individuals 
form  a  close  network  of  fine  interlacing  branches  (pi.  1,  figs.  3 
and  4).  Between  the  two  extremes  in  both  species  are  various 
grades  of  difference,  so  that  the  chromatophores  of  an  unusually 
dark  egg-fed  tadpole  may  not  differ  greatly  from  those  of  a 
lighter  colored  liver-fed  individual. 

A  study  of  sections  shows  that  the  chromatophores  of  the  dark 
tadpoles  are  larger  because  they  contain  many  more  of  the 
brown  melanin  granules.  These  granules  are  so  numerous  that 
the  pigment  forms  a  continuous  network  in  the  intercellular 
spaces,  while  in  the  light  tadpoles  the  amount  of  melanin  is  so 
much  less  that  the  pigment  masses  appear  as  small  spots  with  few 
or  no  processes.  Camera  drawings  of  the  typical  chromatophores 
of  the  two  sorts  make  this  difference  clear. 


Fig.  l 


Fig.  2 


Sections  of  epidermis  of  larvae  of  Sana  sp.     X   450.     (Drawn  with  aid 
of  camera  lucida.) 

Fig.  A.     Tadpole  fed  on  yolk  of  egg. 
Fig.  B.     Tadpole  fed  on  liver. 


82 


University  of  California  Publications  in  Zoology    [VOL.  11 


I.  EFFECT  OF  CHANGES  IN  LIGHT,  HEAT,  AND  FOOD  UPON 
PIGMENTATION 

To  determine  the  influence  of  light  and  heat  upon  the  color 
differences  produced  in  the  tadpoles  by  different  foods,  the  fol- 
lowing experiment  was  carried  out.  Eight  dishes  of  tadpoles, 
all  from  the  same  bunch  of  eggs,  were  arranged  as  shown  below. 


Warm : 

In  constant  temperature,  box  (of 
glass)  kept  at  about  26.5  C. 


Cold: 

In  north  basement  room.  (The 
temperature  of  this  room  varied 
very  little  and  was  always  con- 
siderably below  ordinary  room 
temperature.) 


In  dark. 

(in  black  paste- 
board box) 

In  light. 

In  dark. 

(in  black  paste- 
board box) 

In  light. 


a.  fed  on  liver 
Z>.  fed  on  yolk 

'"c.  fed  on  liver 
d.  fed  on  yolk 

re.  fed  on  liver 
/'.  fed  on  yolk 


ff. 

I"- 


fed  on  liver 
fed  on  yolk 


TABLE  16 

Length  measurements  of  Sana  tadpoles  under  different  conditions  of  light, 
temperature,  and  food.  Measurements  are  average  length  of  all  the 
tadpoles  in  a  dish.  Experiment  began  with  eight  tadpoles  in  each  lot. 


Eana.    Experiment  started  Feb.  10,  1912 


March  5 


\_/UAlU.l 

MUU0    VA    tllC    f.VjM 

;iiuieiit 

in  mm. 

living 

Temperature 

Lighting 

Food 

Warm 

Dark 

Liver 

28.0 

3 

Warm 

Dark 

Egg  yolk 

27.5 

5 

Warm 

Light 

Liver 

31.4 

4 

Warm 

Light 

Egg  yolk 

26.5 

2 

Cold 

Dark 

Liver 

19.3 

8 

Cold 

Dark 

Egg  yolk 

20.8 

8 

Cold 

Light 

Liver 

21.2 

8 

Cold 

Light 

Egg  yolk 

20.3 

8 

AVERAGES 


Cold    . 
Warm 


Light 
Dark 


Egg  yolk 
Liver 


20.4 
28.6 
23.2 
23.0 
22.6 
23.2 


32 

14 
22 
24 
23 
23 


1913]     Johnson:  Pigment  Formation  in  Amphibian  Larvae         83 

The  series  of  colors  is  the  same  under  the  different  conditions 
of  temperature.  In  both  cases  the  series  from  darkest  to  lightest 
coloring  is  as  follows : 

Darkest  tadpoles — Those  fed  upon  liver  and  kept  in  the  light. 

Those  fed  upon  egg  and  kept  in  the  light. 

Those  fed  upon  liver  and  kept  in  the  dark. 
Lightest  tadpoles — Those  fed  upon  egg  and  kept  in  the  dark. 

It  is  plain  from  this  experiment  that  tadpoles  raised  in  the  light 
are  darker  than  those  raised  in  the  dark  and  that  tadpoles  fed 
upon  liver  and  kept  either  in  the  light  or  in  the  dark  are  darker 
than  those  fed  upon  egg  under  the  same  conditions.  The  differ- 
ence in  size  and  development  between  the  tadpoles  kept  at  a  high 
temperature  and  those  in  the  low  temperature  are  so  great  that 
the  two  series  cannot  well  be  compared,  but  no  marked  difference 
in  color  between  the  two  series  can  be  distinguished.  It  is 
evident  that  changes  in  both  food  and  light  influence  the  rate  of 
pigment  formation  markedly  and  that  light  is  a  somewhat 
more  potent  factor  than  liver  in  increasing  the  rate  of  pigment 
production. 

These  conclusions  as  to  increase  of  pigmentation  in  the  light 
and  increase  in  the  rate  of  growth  at  a  higher  temperature  con- 
firm results  already  reported  (table  16).  The  average  length 
measurements  of  all  tadpoles  kept  in  the  dark  compared  with 
those  kept  in  the  light  are  interesting  in  view  of  the  conflicting 
statements  that  have  been  made  by  various  observers  as  to  the 
comparative  rate  of  growth  in  the  dark  and  in  the  light.  These 
figures  support  the  contention  that  there  is  very  little  if  any 
difference  between  the  amount  of  growth  in  the  light  and  in  the 
dark. 

J.  SUMMARY 

A  consideration  of  Weismann's  theory,  the  factor  hypothesis, 
and  the  results  of  chemical  research,  leads  one,  it  seems  to  me, 
to  see  in  the  last  two  named  the  greatest  hope  for  the  solution 
of  problems  of  color  differentiation.  The  difficulties  and  com- 
plications are  great,  but  the  field  for  research  is  correspondingly 
large  and  fruitful. 


84  University  of  California  Publications  in  Zoology    [VOL.  11 

The  present  research  is  by  no  means  complete,  the  results  so 
far  attained  suggesting  numerous  very  pertinent  points  that 
should  be  investigated  farther.  It  does,  however,  furnish  certain 
definite  facts  which  it  is  hoped  will  add  something  to  current 
conceptions  of  color  differentiation. 

The  research  shows  that  pigment  in  Rana  and  Hyla  tadpoles 
is  not  correlated  with  amount  of  nutrition,  as  claimed  by  Tornier 
for  Pelobates  larvae,  but,  as  suggested  by  instances  cited  by 
Darwin  and  Wallace,  is  dependent  rather  upon  substances 
specific  for  color  in  the  nutritive  material.  These  substances  may 
bring  about  a  change  in  the  amount  of  pigment-forming  sub- 
stances produced  or  slightly  alter  the  character  or  combination 
of  these  substances  and  thus  change  the  amount  or  color  of  the 
pigment. 

The  fact  is  made  clear  that  lecithin  acts  as  a  partial  inhibitor 
of  the  tyrosinase  reaction  in  the  test  tube,  and  when  it  is  fed 
to  tadpoles,  pigment  formation  is  checked  to  a  noticeable  degree, 
indicating  that  inhibitors  or  modifiers  of  the  pigment  formation 
may  be  introduced  into  the  organism  with  the  food. 

The  similarity  of  the  effect  of  lecithin  in  the  test  tube  and  in 
the  body  of  the  tadpole  makes  it  probable  that  the  tyrosinase 
reaction  or  a  similar  oxidase  reaction  is  the  basis  of  pigment 
formation  in  the  tadpole. 


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1901.  Zur  Kenntniss  der  Bindung  des  Schwefels  in  den  Proteinstoffen. 
Zeit.  physiol.  Chem.,  34,  207-338. 

OSBORNE,  T.   B.,  AND  CLAPP,  S.   H. 

1906.  The  chemistry  of  the  protein  bodies  of  the  wheat  kernel.    Part 

III.    Am.  Jour.  Physiol.,  17,  231-265. 
PHISALIX,  C. 

1905.     Sur   le   changement   de   coloration   des   larves    de  Phyllodromia 

germanica.     C.R.  Soc.  Biol.,  Paris,  59,  17-18. 
PLIMMER,  R.  H.  A. 

1908.  The  chemical  constitution  of  the  proteins.    (London,  Longmans), 

vol.  i,  xii  +  100  pp.,  vol.  ii,  xi  -f-  66  pp. 
POWERS,  J.  H. 

1908.     Morphological  variation  and  its  causes  in  Amblystoma  tigrinum. 

Studies  from  Zool.  Lab.  Univ.  Nebraska,  71,  77,  9  pis. 
RIDDLE,  O. 

1908.  The  genesis  of  fault-bars  in  feathers  and  the  cause  of  alterna- 

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1909.  Our  knowledge  of  melanin   formation   and  its  bearing  on   the 

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66-67. 


EXPLANATION  OF  PLATE   1 

Fig.  1.     Sana  sp.     The  lighter  tadpole  was  fed  upon  yolk  of  egg,  the 
darker  one  was  fed  upon  liver.     From  photograph,  natural  size. 

Fig.  2.     Hyla  regilla.     The  lighter  tadpole  was  fed  upon  yolk  of  egg, 
the  darker  one  was  fed  upon  liver.    From  photograph,  natural  size. 

Figs.   3-8.     Epidermal   chromatophores   of   amphibian   larvae.      From 
photographs,  magnified  about  forty-five  diameters. 
Fig.  3.     Hyla  regilla.     Tadpole  fed  on  liver. 
Fig.  4.     Hyla  regilla.     Tadpole  fed  on  yolk  of  egg. 
Fig.  5.     Bana  sp.     Chromatophores  of  tail  region,  tadpole  fed  on 

liver. 
Fig.  6.     Bana  sp.     Chromatophores  of  tail  region,  tadpole  fed  on 

yolk  of  egg. 
Fig.  7.     Bana  sp.     Chromatophores  of  body  of  larva,  tadpole  fed 

on  liver. 

Fig.  8.     Bana  sp.     Chromatophores  of  body  of  larva,  tadpole  fed 
on  yolk  of  egg. 


[88] 


UNIV.  CALIF.  PUBL  ZOOL  VOL  II. 


IJOHNSON]  PLATE  I. 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS— (Continued) 

3.  (XXV)  The  Ophiurans  of  the  San  Diego  Region,  by  J.  F.  McClon- 

don.    Pp.  33-64,  plates  1-6.    July,  1909.._ .30 

4.  (XXVI)  Halocynthia  johnsoni  n.  sp.:    A  comprehensive  inquiry  aa  to 

the  extent  of  law  and  order  that  prevails  in  a  single  animal  species, 

by  Wm.  B.  Ritter.     Pp.  65-114,  plates  7-14.    November,  1909 .50 

5.  (XXVII)  Three  Species  of  Cerianthus  from  Southern  California,  by 

H.  B.  Torrey  and  F.  L.  Kleeberger.     Pp.   115-125,   4   text-figure*. 
December,  1909  __ _      .10 

6.  The  Life  History  of   Trypanosoma    dimorphon     Button    &    Todd,    by 

Edward  Hindle.    Pp.  127-144,  plates  15-17,  1  text-figure.    December, 
1909 _ _       450 

7.  (XXVU3)    A  Quantitative   Study  of  the  Development  of  the  Salpa 

Chain  in   Salpa   fusifonnis-runcinaia,  by  Myrtle  Elizabeth  Johnson. 

Pp.  145-176.    March,  1910  .._ .._ .85 

8.  A  Revision  of  the  Genus  Ceratocorys,  Based  on  Skeletal  Morphology, 

by  Charles  Atwood  Kofoid.    Pp.  177-187.    May,  1910 _      40 

9.  (XXIX)  Preliminary  Report  on  the  Hydrographic  Work  Carried  on  by 

the  Marine  Biological  Station  of  San  Diego,  by  George  F.  McEwen. 

Pp.  189-204;  text-figure  and  map.    May,  1910  . ..      .15 

10.  (XXX)  Biological  Studies  on  Corymcrpha.    HI.  Regeneration  of  Hy- 

dranth  and  Holdfast,  by  Harry  Beal  Torrey.    Pp.  205-221;  16  text- 
figures. 

11.  (XXXI)    Note  on  Geotropism  in  Corymorpha,  by  Harry  Beal  Torrey. 

Pp.  223-224;  1  text-figure. 

Nos.  10  and  11  in  one  cover.    August,  1910 ...      .20 

12.  The  Cyclostomatous  Bryosoa  of  the  West  Coast  of  North  America,  by 

Alice  Robertson.    Pp.  225-284;  plates  18-25.    December,  1910 .60 

13.  Significance  of  White  Markings  in  Birds  of  the  Order  Passerlfonnes, 

by  Henry  Chester  Tracy.    Pp.  285-312.    December,  1910 £5 

14.  (XXXIII)  Third  Report  on  the  Copepoda  of  the  San  Diego  Region,  by 

Calvin  Olin  Esterly.    Pp.  313-352;  plates  26-32.    February,  1911 .40 

15.  The  Genus  Gyrocotyle,  and  Its  Significance  for  Problems  of  Cestcde 

Structure  and  Phylogeny,  by  Edna  Earl  Watson.    Pp.  353-468;  plates 
33-48.    June,  1911  _ „ _ 1.00 

VoL,7.     (Contributions  from  the  Museum  of  Vertebrate  Zoology.) 

1.  Two  New  Owls  from  Arizona,  with  Description  of  the  Juvenal  Plum- 

age of  Strix  occidentalis  occidentalis  (Xantus),  by  Harry  S.  Swarth. 

Pp.  1-8.    May,  1910 _      JO 

2.  Birds  and  Mammals  of  the  1909  Alexander  Alaska  Expedition,   by 

Harry  S.  Swarth.  Pp.  9-172;  plates  1-6;  3  text-figures.   January,  1911.    1.50 

3.  An  Apparent  Hybrid  in  the  Genus  Dendroica,  by  Walter  P.  Taylor. 

Pp.  173-177.    February,  1911  „ _ - _ -       .05 

4.  The  Linnet  of  the  Hawaiian  Islands:   a  Problem  in  Speciation,  by 

Joseph  Grinnell.    Pp.  179-195.    February,  1911  _ .15 

5.  The  Modesto  Song  Sparrow,  by  Joseph  Grinnell.    Pp.  J.97-199.    Feb- 

ruary, 1911  _ -       .05 

6.  Two  New  Species  of  Marmots  from  Northwestern  America,  by  H.  S. 

Swarth.    Pp.  201-204.    February,  1911  _ M 

7.  Mammals  of  the  Alexander  Nevada  Expedition  of  1909,  by  Walter  P. 

Taylor.     Pp.  205-307.  June,  1911 1.00 

8.  Description  of  a  New  Spotted  Towhee  from  the  Great  Basin,  by  J. 

Grinnell.     Pp.  309-311.     August,  1911  ...„ ..      .06 

9.  Description  of  a  New  Hairy  Woodpecker  from  Southeastern  Alaska,  by 

H.  S.  Swarth.     Pp.  313-318.     October,  1911  05 

10.  Field  Notes  on  Amphibians,  Reptiles  and  Birds  of  Northern  Humboldt 
County,  Nevada,  with  a  Discussion  of  Some  of  the  Fauna!  Features 
of  the  Region,  by  Walter  P.  Taylor.  Pp.  319-436,  plates  7-12. 
February,  1912  1.00 

VoL  8.    1.  The  Vertical  Distribution   of  Eucalanus  elongatus  in  the  San  Diego 

Region  during  1909,  by  Calvin  O.  Esterly.    Pp.  1-7.    May.  1911. —      .10 

2.  New  and  Rare  Fishes  from  Southern    California,    by   Edwin   Chapin 

Starks  and  William  M.  Mann.     Pp.  9-19,  2  text-figures.     July,  1911      .10 

3.  Classification  and  Vertical  Distribution  of  the  Chaetognatha  of  the  San 

Diego  Region,  Including  Redescriptions  of  Some  Doubtful  Species  of 

the  Group,  by  Ellis  L.  Michael.    Pp.  21-186,  pis.  1-8.    December,  1911    1.75 

4.  Dinoflagellata  of  the  San  Diego  Region,  IV.  The  Genus  Gonyaulax,  with 

Notes  on  Its  Skeletal  Morphology  and  a  Discussion  of  Its  Generic 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS—  (Continued) 

and  Specific  Characters,  by  Charles  Atwood  Kofoid.     Pp.  187-286, 
plates  9-17. 

5.  On  the  Skeletal  Morphology  of  Gonyaulax  catenata   (Levander),  by 

Charles  Atwood  Kofoid.    Pp.  287-294,  plate  18. 

6.  Dinoflagellata  of  the  San  Diego  Region,  V.  On  Spiraulax.  a  New  Qenus 

of  the  Peridinida,  by  Charles  Atwood  Kofoid.    Pp.  295-300,  plate  19. 

Nos.  4,  5,  and  6  in  one  cover.    September,  1911  1.60 

7.  Notes  on  Some  Cephalopods  in  the  Collection  of  the  University  of  Cal- 

ifornia, by  S.  S.  Berry.    Pp.  301-310,  plates  20-21.    September,  1911      J.O 

8.  On  a  Self-closing  Plankton  Net  for  Horizontal  Towing,  by  Charles  At- 

wood Kofoid.    Pp.  311-348,  plates  22-25. 

D.  On  an  Improved  Form  of  Self-closing  Water-bucket  for  Plankton  In- 
vestigations, by  Charles  Atwood  Kofoid.    Pp.  349-352. 

Nos.  8  and  9  in  one  cover.    November  18,  1911  „ .40 

1.  The  Horned  Lizards  of  California  and  Nevada  of  the  Genera  Phryno- 

soma  and  Anota,  by  Harold  C.  Bryant.    Pp.  1-84,  pis.  1-9.    December, 
1911    .75 

2.  On  a  Lymphoid  Structure  Lying  Over  the  Myelencephalon  of  Lcpisos- 

tfis,  by  Asa  C.  Chandler.  Pp.  85-104,  plates  10-12.  December,  1911  .25 
S.  Studies  on  Early  Stages  of  Development  in  Rats  and  Mice,  No.  S,  by 
£.  L.  Mark  and  J.  A.  Long.  The  Living  Eggs  of  Rats  and  Mice  with 
a  Description  of  Apparatus  for  Obtaining  and  Observing  Them  (Pre- 
liminary paper),  by  J.  A.  Long.  Pp.  105-136,  plates  13-17.  February, 
1912 _ .30 

4.  The  Marine  Biological  Station  of  San  Diego,  Its  History,  Present  Con- 

ditions, Achievements,  and  Aims,  by  Wm.  E.  Ritter.    Pp.  137-248, 

pis.  18-24,  and  2  maps.    March,  1912 _ 1.00 

5.  Oxygen  and  Polarity  In  Tubularia,  by  Harry  Beal  Torrey.    Pp.  249- 

251.    May,  1912 .05 

6.  The  Occurrence  and  Vertical  Distribution  of  the  Copepoda  of  the  San 

Diego  Region,  with  particular  reference  to  Nineteen  Species,  by  Cal- 
vin O.  Esterly.    Pp.  253-340,  7  text-figures.    July,  1912 1.00 

7.  Observations  on  the  Suckling  Period  in  the  Guinea-pig,  by  J.  Marion 

Read.    Pp.  341-351.    September,  1912  10 

8.  Haeckel's  Sethocephalus  Eucecryphalus  (Radiolaria)  a  Marine  Ciliate,  by 

Charles  Atwood  Kofoid.    Pp.  353-357.    September,  1912 ~ 05 

(Contributions  from  the  Museum  of  Vertebrate  Zoology.) 

1.  Report  on  a  Collection  of  Birds  and  Mammals  from  Vancouver  Island, 

by  Harry  S.  Swarth.    Pp.  1-124,  plates  1-4.    February,  1912 1.00 

2.  A  New  Cony  from  the  Vicinity  of  Mount  Whitney,  by  Joseph  Grinnell. 

Pp.  125-129.     January,  1912  05 

S.  The  Mole  of  Southern  California,  by  J.  Grinnell  and  H.  S.  Swarth. 
Pp.  131-136,  2  text-figures. 

4.  Myotis  orinotnus  Elliot,  a  Bat  New  to  California,  by  J.  Grinnell  and 

H.  S.  Swarth.    Pp.  137-142,  2  text-figures. 

Nos.  3  and  4  in  one  cover.    April,  1912 12 

5.  The  Bighorn  of  the  Sierra  Nevada,  by  Joseph  Grinnell.    Pp.  143-153, 

4  text-figures.    May,  1912 ~ JLO 

6.  A  New  Perognatlms   from  the  San   Joaquin   Valley,   California,   by 

Walter  P.  Taylor.    Pp.  155-166,  1  text-figure. 

7.  The  Beaver  of  West  Central  California,  by  Walter  P.  Taylor.     Pp. 

167-169. 

Nos.  6  and  7  in  one  cover.    May,  1912  15 

8.  The  Two  Pocket  Gophers  of  the  Region  Contiguous  to  the  Lower  Colo- 

rado River,  in  California  and  Arizona,  by  Joseph  Grinnell,    Pp.  171- 

178.    June,  1912 15 

9.  The   Species  of  the  Mammalian  Genus  Sorex  of  West-Central   Cali- 

fornia, with  a  note  on  the  Vertebrate  Palustrine  Faunas  of  the 
Region,  by  Joseph  Grinnell.    Pp.  179-195,  figs.  1-6.    March,  1913 15 

1.  Birds  in  Relation  to  a  Grasshopper  Outbreak  in  California,  by  Harold 

C.  Bryant.    Pp.  1-20.    November,  1912  20 

2.  On  the  Structure  and  Relationships  of  Dinosphacra  Palustris  (Lemm.), 

by  Charles  Atwood  Kofoid  and  Josephine  Rigden  Michener.    Pp.  21- 

28.    December,  1912  10 

3.  A    Study   of   Epithelioma   Contagiosum    of   the   Common   Fowl,   by 

Clifford  D.  Sweet.    Pp.  29-51.    January,  1913  25 

4.  The  Control  of  Pigment  Formation  in  Amphibian  Larvae,  by  Myrtle 

E.  Johnson.    Pp.  53-88,  plate  1.    March,  1913 35 


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